The interaction between T cell immunoglobulin-and mucin-domain-containing molecule (Tim-3) expressed on T helper 1 (Th1) cells, and its ligand, galectin-9, negatively regulates Th1-mediated immune responses. However, it is poorly understood if and how the Tim-3/ galectin-9 signaling pathway is involved in immune escape in patients with hepatocellular carcinoma (HCC). Here we studied the expression, function, and regulation of the Tim-3/ galectin-9 pathway in patients with hepatitis B virus (HBV)-associated HCC. We detected different levels of galectin-9 expression on antigen-presenting cell (APC) subsets including Kupffer cells (KCs), myeloid dendritic cells (DCs), and plasmacytoid DCs in HCC. The highest galectin-9 expression was on KCs in HCC islets, not in the adjacent tissues. Furthermore, Tim-3 expression was increased on CD4 1 and CD8 1 T cells in HCC as compared to the adjacent tissues, and Tim-3 1 T cells were replicative senescent and expressed surface and genetic markers for senescence. Interestingly, tumor-infiltrating T-cell-derived interferon (IFN)-c stimulated the expression of galectin-9 on APCs in the HCC microenvironment. Immunofluorescence staining revealed a colocalization of Tim-3 1 T cells and galectin-91 KCs in HCC. Functional studies demonstrated that blockade of the Tim-3/ galectin-9 signaling pathway importantly increased the functionality of tumor-infiltrating Tim-3 1 T cells as shown by increased T-cell proliferation and effector cytokine production. Finally, we show that the numbers of Tim-3 1 tumor-infiltrating cells were negatively associated with patient survival. Conclusion: Our work demonstrates that the Tim-3/galectin-9 signaling pathway mediates T-cell senescence in HBV-associated HCC. The data suggest that this pathway could be an immunotherapeutic target in patients with HBV-associated HCC. (HEPATOLOGY 2012;56:1342-1351 H epatocellular carcinoma (HCC) is one of the most common cancers. More than 80% of patients are not candidates for curative treatments with the final diagnosis, and are linked to chronic infection with the hepatitis B (HBV) or hepatitis C (HCV) viruses based on different regions.